Aptamers are nucleic acid molecules having highly specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing.
Aptamers, like peptides generated by phage display or monoclonal antibodies (“mAbs”), are capable of specifically binding to selected targets and modulating the target's activity, e.g., through binding aptamers may block their target's ability to function. Created by an in vitro selection process from pools of random sequence oligonucleotides, aptamers have been generated for over 100 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind other proteins from the same gene family). A series of structural studies have shown that aptamers are capable of using the same types of binding interactions (e.g., hydrogen bonding, electrostatic complementarities, hydrophobic contacts, steric exclusion) that drive affinity and high selective binding in antibody-antigen complexes.
Aptamers have a number of desirable characteristics for use as therapeutics and diagnostics including high selectivity and affinity, biological efficacy, and excellent pharmacokinetic properties. In addition, they offer specific competitive advantages over antibodies and other protein biologics, for example:
1) Speed and control. Aptamers are produced by an entirely in vitro process, allowing for the rapid generation of initial leads, including therapeutic leads. In vitro selection allows the selectivity and affinity of the aptamer to be tightly controlled and allows the generation of leads, including leads against both toxic and non-immunogenic targets.
2) Toxicity and Immunogenicity. Aptamers as a class have demonstrated therapeutically acceptable toxicity and lack of immunogenicity. In chronic dosing of rats or woodchucks with high levels of aptamer (10 mg/kg daily for 90 days), no toxicity is observed by any clinical, cellular, or biochemical measure. Whereas the efficacy of many monoclonal antibodies can be severely limited by immune response to antibodies themselves, it is extremely difficult to elicit antibodies to aptamers most likely because aptamers cannot be presented by T-cells via the MHC and the immune response is generally trained not to recognize nucleic acid fragments.
3) Administration. Whereas most currently approved antibody therapeutics are administered by intravenous infusion (typically over 24 hours), aptamers can be administered by subcutaneous injection (aptamer bioavailability via subcutaneous administration is >80% in monkey studies (Tucker et al., J. Chromatography B. 732: 203-212, 1999)). This difference is primarily due to the comparatively low solubility and thus large volumes necessary for most therapeutic mAbs. With good solubility (>150 mg/mL) and comparatively low molecular weight (aptamer: 10-50 kDa; antibody: 150 kDa), a weekly dose of aptamer may be delivered by injection in a volume of less than 0.5 mL. In addition, the small size of aptamers allows them to penetrate into areas of conformational constrictions that do not allow for antibodies or antibody fragments to penetrate, presenting yet another advantage of aptamer-based therapeutics or prophylaxis.
4) Scalability and cost. Therapeutic aptamers are chemically synthesized and consequently can be readily scaled as needed to meet production demand. Whereas difficulties in scaling production are currently limiting the availability of some biologics and the capital cost of a large-scale protein production plant is enormous, a single large-scale oligonucleotide synthesizer can produce upwards of 100 kg/year and requires a relatively modest initial investment.
5) Stability. Therapeutic aptamers are chemically robust. They are intrinsically adapted to regain activity following exposure to factors such as heat and denaturants and can be stored for extended periods (>1 yr) at room temperature as lyophilized powders.
Thrombin
Thrombin is a multifunctional serine protease that has procoagulant and anticoagulant activities. As a procoagulant enzyme, thrombin clots fibrinogen, activates clotting factors V, VIII, and XIII, and activates platelets. The specific cleavage of fibrinogen by thrombin initiates the polymerization of fibrin monomers, a primary event in blood clot formation. The central event in the formation of platelet thrombi is the activation of platelets from the “nonbinding” to the “binding” mode. Thrombin is a physiologic activator of platelet aggregation. Thus, as a procoagulant, thrombin plays a key role in the arrest of bleeding (physiologic hemostasis) and formation of vaso-occlusive thrombi (pathologic thrombosis).
As an anticoagulant thrombin binds to thrombomodulin (TM), a glycoprotein expressed on the surface of vascular endothelial cells. TM alters substrate specificity from fibrinogen and platelets to protein C through a combination of an allosteric change in the active site conformation and an overlap of the TM and fibrinogen binding sites on thrombin. Activated protein C, in the presence of a phospholipid surface, Ca2+, and a second vitamin K-dependent protein cofactor, protein S, inhibits coagulation by proteolytically degrading factors Va and VIIIa. Thus, the formation of the thrombin-TM complex converts thrombin from a procoagulant to an anticoagulant enzyme, and the normal balance between these opposing activities is critical to the regulation of hemostasis.
Coagulation Disorders
Vascular injury and thrombus formation represent the key events in the pathogenesis of various vascular diseases, including atherosclerosis. The pathogenic processes of the activation of platelets and/or the clotting system, leading to thrombosis in various disease states and in various sites, such as the coronary arteries, cardiac chambers, and prosthetic heart valves, appear to be different. Therefore, the use of a platelet inhibitor, an anticoagulant, or a combination of both may be required in conjunction with thrombolytics to open closed vessels and prevent reocclusion.
Controlled proteolysis by compounds of the coagulation cascade is critical for hemostasis. As a result, a variety of complex regulatory systems exist that are based, in part, on a series of highly specific protease inhibitors. In a pathological situation functional inhibitory activity can be interrupted by excessive production of active protease or inactivation of inhibitory activity. Perpetuation of inflammation in response to multiple trauma (tissue damage) or infection (sepsis) depends on proteolytic enzymes, both of plasma cascade systems, including thrombin, and of lysosomal origin. Multiple organ failure (MOF) in these cases is enhanced by the concurrently arising imbalance between proteases and their inhibitory regulators. Furthermore, an imbalance of thrombin activity in the brain may lead to neurodegenerative diseases.
Coronary Artery Bypass Graft (CABG) Surgery
In 2001, the American Heart Association reported that an estimated 12.4M patients in the U.S. were diagnosed with some form of coronary artery disease. Given thrombin's importance in the coagulation process, an anti-thrombin agent or an agent that decreases or inhibits thrombin activity is the anticoagulant used, e.g., during coronary artery bypass graft (hereinafter “CABG”) surgery, percutaneous coronary intervention (hereinafter “PCI”) and acute coronary syndrome. As of 2001, more than 570,000 CABG procedures were performed annually in the U.S. and it is estimated that over 700,000 procedures are performed worldwide. Currently, the most commonly used anticoagulant is heparin which must be used with the antidote protamine. However, heparin-protamine treatment is associated with a number of serious side-effects including bleeding and thrombocytopenia (platelet count reduction) which is often asymptomatic but may be associated with life-threatening arterial or venous thrombosis. In addition, heparin-protamine treatment has a number of other disadvantages including: non-specific binding to plasma proteins which results in resistance in some patients; heparin cannot inhibit clot-bound thrombin; heparin has non-linear kinetics making dosing difficult to control; and heparin is manufactured from beef or pork tissues which have an inherent safety risk arising from the possibility for transmission of viruses and/or prions. Consequently, a number of newer, higher-cost anticoagulants, such as low molecular weight heparins and Angiomax®, have gained significant penetration into this market. However, these compounds have similar side-effects and their anticoagulation activity cannot be reversed rapidly.
Thus, there is a significant unmet medical need for a safe, moderate-cost anticoagulant that does not require a separate reversing agent and which is not associated with the side effects and disadvantages listed above. Accordingly, it would be beneficial to have agents that decrease or inhibit the activity of thrombin for use as therapeutics in the treatment of coagulation-related disorders.